The bipolymer glycogen is the major storage form of glucose as an energy source in the body. Regulation of the enzyme, glycogen phosphorylase (GP), modulates the catabolic breakdown of glycogen in various tissues and in distinct stages of cell development by mobilizing glucose reserves for ATP energy production. GP is under a complex set of controls that prevent glycogen breakdown except when ATP is needed. The breakdown of glycogen involves substrates, intermediates, cofactors, activators, inhibitors, enzymatic interconversions and hormonal interactions. When blood glucose is low, GP is activated and causes muscle and liver glycogen to be broken down to yield glucose. The rate of glucose mobilization by this system is regulated by a cascade of kinase reactions initiated by epinephrine and an increase of calcium ions in the cytosol. Glucose produced from glycogen in muscle cells is oxidized quickly to CO.sub.2 yielding ATP as a source of energy for muscle contraction. Glucose generated from liver glycogen is secreted into the blood where it serves as an energy source for many body tissues that do not store energy reserves, such as the brain.
Glycogen phosphorylase exists in active (phosphorylase a) and inactive (phosphorylase b) forms, the ratio of which is controlled by the rate of interconversion between the different forms and affects the rate of conversion of glycogen into glucose. Activation occurs through the phosphorylation of GP at specific serine residues by the enzyme, phosphorylase kinase (Phk), a Ca.sup.2+ dependent enzyme. The activity of GP (hence the catabolic breakdown of glycogen) therefore depends upon its phosphorylation by Phk.
Glycogen phosphorylase, and its activation through phosphorylase kinase (Phk), is situated at a strategically important point between the fuel reservoir, glycogen, and the glycolytic pathways that provide glucose as fuel for various cellular activities underlying tissue maintenance and development. Heritable deficiencies in Phk underlie a remarkably heterogeneous group of glycogen utilization disorders in humans. At least ten different forms of Phk deficiency have been described (Schneider A et al (993) Nature Gene 5: 381-385).
Phk is one of the largest and most complex of the known protein kinases. The enzyme has a quaternary structure based upon 4 subunits: alpha, beta, gamma, and delta. Phosphorylase kinase exists in an active phosphorylated form and an inactive dephosphorylated form. The regulatory alpha and beta subunits are phosphorylated by cAMP-dependent protein kinase. The delta subunit is calmodulin and mediates regulation by Ca.sup.2+. The binding of Ca.sup.2+ to the delta subunit activates the complex, which is maximally active if at least the alpha subunit is phosphorylated. The N-terminal region of the gamma subunit contains the catalytic protein kinase domain, within approximately residues 1-300, while the C-terminal region (approximately residues 300-385) contains two of the important calmodulin-binding domains required to activate the protein complex (Huang C Y, et al(1995) J Biol Chem 270(13): 7183-7188). Within this subunit, residues Glu(E)111 and Glu(E) 154 are purported to be important for substrate binding (Huang et al, supra) and residues Asn(N) 155, Asp(D) 168, Phe(F)169, and Gly(G) 170 are important for catalytic activity (Huang C Y et al (1994)Biochem 33(19:5877-83). Mutations affecting different subunits, especially the catalytic gamma subunit, are expected to contribute to genetic heterogeneity (Wehner M et al (1994) Hum Mol Genet 3(11):1983-87). Two isoforms, each encoded by separate genes, are currently known for the gamma subunit in muscle and testis (Calalb MB et al(1992) J Biol Chem 267:1455-63).
Phosphorylase kinase deficiency has significant potential for genetic heterogeneity, since the enzyme is composed of four non-identical subunits. Inborn genetic errors affecting glycogen breakdown, namely deficiencies of phosphorylase kinase (Phk), have been reviewed (Moses SW (1990) J Inherit Metab Dis 13(4):452-65). Moreover, deficiency of glycogen catabolic functions have been frequently reported in families as well as in reports of different enzyme defects among siblings. Mutations affecting different subunits and isoforms of Phk are expected to contribute to this genetic heterogeneity (Wehner M et al, supra).
Muscle glycogenosis caused by Phk deficiency leads to exercise intolerance, weakness and muscular atrophy. Hepatic Phk deficiency is also associated with certain hepatic diseases characterized by hypoglycemia and hepatomegaly. Severe muscular (myopathic) forms of Phk deficiency have been reported which start in infancy, as well as a late onset form, and are characterized by exercise intolerance and muscle cramps. Another autosomal recessively inherited variant in which both liver and muscle enzymes were affected has been described separately in two families (Lederer 3 et al (1980) Biochem Biophys Res Commun 92:169-174; Basnan N et al (1981) Pediatr Res 15:299). Experts have frequently suggested that genes which are responsible for these conditions, as well as ones for enzyme replacement therapy, should be sought (Wehner M et al (1995) Hum Genet 96(5):616-618)
No primary therapy for the Phk enzyme deficiency is presently available in any of the glycogenoses. The main therapeutic efforts are dietary in nature. Patients with enzyme deficiencies have in their early years limited fasting tolerances. These characteristics of hypoglycemia are addressed with frequent feedings, nocturnal gastric infusions of glucose and uncooked cornstarches. High protein diets have also been advocated but have not been shown to prevent the course of disease, especially in infantile forms. Most infantile cases are lethal within two years (Moses SW (1990) J Inherit Metab Dis 13(4):452-65).
It is therefore clear that there continues to be a significant longfelt need for agents which are useful for the detection, treatment, and correction of pathophysiological conditions caused by aberrant forms of the Phk and by deficiencies in Phk activity.